This research program deals with basic mechanisms of transcription and with transcriptional regulation of the activities of genes. Its implications for human health relate to the development application of precise and specific interventions in gene expression that are based on a broad and fundamental understanding of transcription mechanisms as well as their associated protein-protein and protein-nucleic acid interactions. The proposed experiments deal with two transcription systems. Projects dealing with eukaryotic nuclear RNA polymerase III pursue the following objectives: 1) precisely delineate the major binding site for the Bdp1 subunit of the transcription factor (TF)IIIB; 2) devise a perfectly symmetric (i.e. palindromic) DNA site for TFIIIB; 3) explore new TBP-Brf1 fusion proteins in assembly of transcriptionally functional TFIIIB; 4) probe the structure of transcription initiating promoter complexes and transcript-elongating transcription complexes by RNA protein cross-linking to answer specific questions about the role of the Brf1 subunit of TFIIIB in initiation of transcription, transcript elongation, rapid polymerase recycling and coupling between transcription and post-transcriptional RNA processing; 5) probe the structure of the RNA polymerase III surface by protein-protein cross-linking to answer specific questions about the locations of five specified subunits and about the TFIIIB-polymerase III contact surface; 6) use chromatin immunoprecipitation (CHIP) to determine occupancy of selected RNA polymerase III-transcribed genes by TFIIIB, TFIIIC and RNA polymerase III, as well as kinetics of change of occupancy upon selective disruption of RNA polymerase III transcription; 7) dissect the mechanism that directs DNA transposition by the retrovirus-like Ty3 transposable element to RNA polymerase III promoters by interaction with TFIIIB (in collaboration); 8) analyze RNA polymerase III transcription through obstructing nucleosomes (in collaboration). A parallel group of projects deals with a regulatory system that couples transcription to concurrent genome replication in bacteria. The activator of this regulatory system is a sliding clamp protein; a specific RNA polymerase-bound co-activator is also required. The principal projects pursue the following objectives: 1) determine the mode of attachment of the co-activator to RNA polymerase and associated protein secondary structure changes; 2) determine whether the co-activator functions only in initiation of transcription or whether it remains attached to RNA polymerase throughout transcription; 3) dissect the architecture of this transcription regulatory system and its relationship to the architecture and function of bacterial sigma initiation factors by means of an extensive series of recombinant constructions, targeted mutagenesis and protein domain fusions.